The proposed study will help elucidate whether 5-methylcytosine (m5C) residues in DNA are more likely to be the sites of mutation than are the analogous cytosine (C) residues. Studies aimed at this question have not been reported to date. As mutagenic agents, sodium bisulfite, heat (80 degrees C), and ultraviolet (UV) irradiation will be used. It is predicted that deamination of m5C residues to thymine residues will be a major consequence of treatment with the former two agents and a minor but mutagenically significant consequence of treatment with the latter agent. In all cases the effect of these agents on m5C residues and on C residues in DNA will be compared. A study of the consequences of treating m5C residues in DNA with sodium bisulfite, heat, or UV light will be conducted on phage XP-12 DNA, the only naturally occurring DNA known to have all of its C residues 5-methylated. This DNA will be radioactively labeled in only its m5C residues. Xanthomonas oryzae DNA, which has a similar base composition except that its C residues are not methylated, will be radiolabeled specifically in its C residues and used as the source of normal DNA. After treatment, the labeled bases of the DNAs will be analyzed by chromatography. The relative mutagenic potential of alteration of m5C and of C residues in DNA will be examined after treatment of a specific fragment of phiX-174 DNA with sodium bisulfite or heat. This DNA fragment from the minus strand will have been either extensively substituted with m5C or will contain only unmethylated C residues as normal. After reconstitution of the replicative form of the DNA with the treated fragment, the formation of host range mutant phage will be monitored in transfected, normal or repair-deficient Escherichia coli spheroplasts.